Kinetic studies of conformational changes of macromolecules provide valuable information on the function and dynamics of biomolecules. A thorough study of reaction kinetics requires knowledge of the transient state of molecules during conformational changes. This necessitates the investigation of molecular structure at various time points, as in time-resolved crystallography, often based on pump-probe methods.
In chemical processes, such as a substrate-enzyme interaction, or protein folding or unfolding, mixing of two liquids or solutions initiates a reaction. The fast nature of some conformational changes, e.g. protein folding or unfolding, calls for new experimental methods that access rapid time scales. Several techniques have been adopted in the past, such as photochemical triggering, temperature/pressure jump and rapid fluid mixing.
X-ray Free Electron Lasers (XFELs) have opened up new opportunities for crystallography due to the ability to outrun radiation damage in a “diffract-before-destroy” read-out mode, and may also allow diffraction measurements with very high time-resolution at room temperature where multiple copies of a sample can be provided. XFELs may provide 1012 photons per 50 fs hard-Xray pulse, currently at a pulse repetition rate of 120 Hz. The requirements for sample delivery in XFEL experiments, such as high replenishment rate in a hydrated environment in vacuum thus pose challenges for existing closed cell liquid mixing methods. Turbulent mixing may achieve extremely fast mixing times, but high sample consumption limits its utility for most biological samples. The extremely short and fixed mix-to-probe delay time also limits its application to measure full reaction time courses. Microfluidic devices can usually be ruled out due to the extremely bright XFEL beam, which vaporizes any material in its path.
Accordingly, a need exists for an apparatus and a method for allowing time-resolved spectroscopy for the study of chemical kinetics.